SBIR-STTR Award

Antibodies are a key component of the adaptive immune system, released in response to disease in order to target foreign molecular surfaces. Due to their capacity for high target affinity and specificity, they’ve become one of the fastest growing classes of therapeutic molecules addressing a range of disease including infectious diseases, auto-immune diseases, and cancer. Camelids, including llamas, camels, and alpacas, produce a unique repertoire of antibodies that includes both dual chain antibodies and single chain antibodies. Single chain antibodies, due to their comparative structural simplicity, are simpler to express and develop at the scale required for therapeutics. In addition, single chain antibodies are able to bind to small epitopes, such as enzyme active sites, that would be hidden to larger, dual chain antibodies. The binding domain of the single chain antibody may be small enough to infiltrate traditionally difficult to access tissues, including crossing the blood-brain barrier. Current approaches to single chain antibody discovery require the collection of cells that encode the antibody genes, including memory B cells and plasma cells. Target-specific antibodies are selected after the antibody transcripts are cloned into a display system, such as phage or yeast. While memory B cells and plasma cells represent only a minute fraction of the cells located in peripheral blood, target- specific antibodies are present in high concentration in blood after an infection. Each plasma cell can secrete thousands of antibodies per minute. Digital Proteomics is developing Alicanto, a technology that utilizes the antibodies circulating in blood to identify target-specific antibodies. Alicanto integrates two sources of information about the antibody repertoire. First, Alicanto constructs a database of potential antigen-specific antibodies by performing next-generation sequencing of antibody transcripts. Next, Alicanto enriches for target-specific antibodies from the blood using affinity chromatography and subjects the antibodies to tandem mass spectrometry. Finally, Alicanto uses machine learning models to integrate the sequencing and mass spectrometry data to derive a collection of target-specific antibody candidates. For single chain antibody discovery, Alicanto will use specialized primers and enrichment techniques to isolate only the subset of the antibody repertoire that contains the single chain antibodies. Alicanto will be used to discover high affinity, single chain antibodies for development as therapeutic molecules.

Public Health Relevance Statement:
Antibodies are produced natively in humans to fight disease and the molecules have garnered significant attention due to their therapeutic potential. Single-chain antibodies, which are smaller than conventional antibodies, represent a new frontier for antibody-based therapeutics due to their ability to target molecular surfaces that would be hidden to larger antibodies, as well as the relative simplicity of their development. Digital Proteomics is developing Alicanto to discover single-chain antibodies in llama that can be developed into human therapies.

Project Terms:
Active Sites; Adaptive Immune System; Address; Affinity; Affinity Chromatography; Alpaca; Antibodies; antibody engineering; Antibody Repertoire; Antibody Therapy; Antigen Targeting; Antigens; Aspirate substance; Attention; Autoimmune Diseases; B cell repertoire; B-Lymphocytes; Bacteriophages; Binding; biophysical properties; Blood; Blood - brain barrier anatomy; Bone Marrow; Brain; Camels; Cations; Cell Fraction; Cells; Characteristics; Cloning; Collection; Communicable Diseases; comparative; Data; Databases; design; Development; Diagnostic; digital; dimer; Disease; Drug Kinetics; Enzymes; Epitopes; Exhibits; fighting; frontier; G-substrate; Gel; Genes; Genus staphylococcus; Homo; Human; Immunization; Immunization Schedule; Industry Standard; Infection; Lead; Libraries; Light; Llama; Machine Learning; Malignant Neoplasms; Mass Spectrum Analysis; melting; Memory B-Lymphocyte; Methods; Microbe; Modeling; Molecular; Molecular Target; Monoclonal Antibodies; nanobodies; neglected tropical diseases; next generation sequencing; Organism; Oryctolagus cuniculus; peripheral blood; Pharmacodynamics; Plasma Cells; polypeptide; preference; Proteins; proteogenomics; Proteomics; Protocols documentation; Publishing; response; sample collection; screening; Serum; Solid Neoplasm; Source; Specificity; Spleen; Surface; System; tandem mass spectrometry; Techniques; Technology; Temperature; Therapeutic; therapeutic candidate; Thrombotic Thrombocytopenic Purpura; Time; Tissues; Transcript; transcriptomics; Venoms; Whole Blood; Work; Yeasts

Heavy chain-only antibodies (HCAbs) are a unique class of antibodies only produced in camelids and cartilagenous fish. Unlike conventional antibodies that consist of two heavy chains and two light chains, HCAbs consist only of heavy chains. The antigen-binding portion of the camelid HCAb, called the VHH, is a polypeptide fragment with wide utility across crystallography, imaging, and therapeutics. The small size and comparatively simple structure of the VHH as compared to conventional antibodies makes them economical to produce and structurally stable at a wider pH and temperature range. Growing evidence suggests that the epitopes targeted by HCAbs are distinct from those targeted by conventional antibodies. Concave domains, such as enzyme active sites, are one category that are preferentially targeted by HCAbs than conventional antibodies. This is in part due to the ability of VHHs to produce convex conformations which enables targeting of sites that are inaccessible to conventional antibodies. Phage display is the predominant method for discovering novel VHHs. HCAb transcripts are cloned into phagemids, and phages that express a VHH that binds the target are enriched through the process of panning. Attrition at cloning, panning, or final selection reduces the accessible diversity of the immune system, and delivers antibodies that may not be as diverse as the response mounted by the original host organism. In contrast to display-based methods, Alicanto combines next-generation sequencing and mass spectrometry to directly identify antigen-specific circulating HCAbs from camelids. Assessment of circulating HCAbs in serum is the primary method by which an immunization is determined to be successful. While the serum is discarded after screening in the phage display workflow, Alicanto uses mass spectrometry to identify the individual HCAbs comprising the target-specific circulating antibodies. This enables Alicanto to discover low abundance antibodies against challenging targets such as peptides and small molecules. By constructing an in silico library of HCAb sequences using next-generation sequencing, Alicanto precludes the need to clone into an intermediate host such as phages. Through direct analysis of the immune response of llamas, Alicanto will deliver more, high affinity VHHs for use in research, diagnostics, and therapeutics.

Public Health Relevance Statement:
Single chain antibodies derived from camelids are a unique class of molecules ideally suited for a broad range of applications including therapeutics, structural biology, and histology. Alicanto is a novel platform for discovering single chain antibodies from llamas that rapidly delivers more, high affinity single chain antibodies than currently available technologies. Alicanto will enable broader use of these unique molecules across research, diagnostics, and therapeutics.

Project Terms:
Active Sites; Affinity; Alanine; Animals; Antibodies; Antibody Response; antigen binding; Antigen Targeting; Antigens; B-Lymphocytes; Bacteriophages; base; Behavior; Binding; candidate selection; Categories; Cations; Cities; Cloning; commercial application; comparative; Computer Analysis; Computer software; Computing Methodologies; Crystallography; Custom; Cytometry; Data; Databases; Diagnostic; Engineering; Ensure; Enzyme-Linked Immunosorbent Assay; Enzymes; Epitopes; Escherichia coli; Fishes; Gene Conversion; Genes; Genetic; Goals; head-to-head comparison; Histology; Image; Immune response; Immune system; Immunization; immunological diversity; Immunoprecipitation; in silico; Individual; interest; Keyhole Limpet Hemocyanin; lead candidate; Libraries; Light; Link; Llama; Mass Spectrum Analysis; Methods; Modality; Molecular Conformation; next generation sequencing; novel; Organism; Peptides; Performance; Pesticides; Phage Display; Phase; Phylogenetic Analysis; polypeptide; Process; Proteins; proteogenomics; Reagent; Research; response; RNA; RNA Editing; Sampling Biases; screening; Serum; Site; small molecule; structural biology; Structure; synthetic construct; tandem mass spectrometry; Technology; Temperature; Therapeutic; TimeLine; Transcript; Western Blotting; Work